Reducing myelin-mediated inhibition of axon regeneration

ABSTRACT

Oligodendrocyte-myelin glycoprotein (OMgp)-specific binding agents are used to reduce OMgp-mediated axon growth inhibition. Mixtures of axons and OMgp and mixtures of Nogo receptor (NgR) and OMgp are used in pharmaceutical screens to characterize agents as inhibiting binding of NgR to OMgp and promoting axon regeneration.

This application is a continuation of U.S. Ser. No. 10/127,058 (now U.S.Pat. No. 7,309,485), filed Apr. 19, 2002, which is a continuingapplication of U.S. Ser. No. 10/006,002 filed on Dec. 3, 2001 nowabandoned.

This work was supported by Federal Grant No. 1R21NS41999-01 from NINDS.The government may have rights in any patent issuing on thisapplication.

INTRODUCTION

1. Field of the Invention

The invention is in the field of reducing meylin-mediated inhibition ofaxon regeneration.

2. Background of the Invention

The inhibitory activity associated with myelin is a major obstacle forsuccessful axon regeneration in the adult mammalian central nervoussystem (CNS)^(1,2). In addition to myelin associated glycoprotein(MAG)³⁻⁴ and Nogo-A⁵⁻⁷, evidence suggests the existence of otherinhibitors in CNS myelin⁸. We show that a glycosylphosphatidylinositol(GPI)-anchored CNS myelin protein, oligodendrocyte-myelin glycoprotein(OMgp), is a potent inhibitor of neurite outgrowth. Like Nogo-A, OMgpcontributes significantly to the inhibitory activity associated with CNSmyelin. To elucidate the mechanisms that mediate this inhibitoryactivity of OMgp, we screened an expression library and identified theNogo receptor (NgR)⁹ as a high affinity OMgp binding protein. Cleavageof NgR and other GPI-linked proteins from the cell surface rendersdorsal root ganglion axons insensitive to OMgp. Introduction ofexogenous NgR confers OMgp-responsiveness to otherwise insensitiveneurons. We conclude that OMgp is an physiological neurite outgrowthinhibitor that acts through and is a physiological ligand of the NgR andits associated receptor complex. We show that Interfering with theOMgp/NgR pathway allows lesioned axons to regenerate after injury invivo.

SUMMARY OF THE INVENTION

The invention provides methods and compositions for reducingOMgp-mediated axon growth inhibition. In one embodiment, the methodcomprising steps (a) contacting a mixture comprising an axon andisolated OMgp with an agent and under conditions wherein but for thepresence of the agent, the axon is subject to growth inhibition mediatedby the OMgp; and (b) detecting resultant reduced axon growth inhibition.In an alternative embodiment, the method comprises steps: (a) contactinga mixture comprising an axon and OMgp with an exogenous OMgp-specificbinding agent and under conditions wherein but for the presence of theagent, the axon is subject to growth inhibition mediated by the OMgp,whereby the agent binds the OMgp and reduces the growth inhibition; and(b) detecting resultant reduced axon growth inhibition.

These methods may be practiced with isolated neurons in vitro, or withneurons in situ. Suitable agents include (i) a candidate agent notpreviously characterized to bind OMgp nor reduce axon growth inhibitionmediated by OMgp; (ii) a candidate agent not previously characterized toreduce axon growth inhibition mediated by OMgp; (iii) an OMgp-specificantibody fragment; (iv) a soluble NgR peptide sufficient to specificallybind the OMgp and competitively inhibit binding of the OMgp to NgR; etc.In more particular embodiments, the recited isolated OMgp consistsessentially of OMgp, particularly wherein the OMgp is soluble andGPI-cleaved and/or the OMgp is recombinantly expressed on a surface of acell.

The invention also provides methods and compositions for characterizingan agent as inhibiting binding of NgR to OMgp. In one embodiment, thismethod comprising the steps (a) incubating a mixture comprising NgR,OMgp and an agent under conditions whereby but for the presence of theagent, the NgR and OMgp exhibit a control binding; and (b) detecting areduced binding of the NgR to the OMgp, indicating that the agentinhibits binding of the NgR to the OMgp.

The method may be practiced in a variety of alternative embodiments,such as (i) wherein at least one of the NgR and OMgp is soluble andGPI-cleaved; (ii) wherein one of the NgR and OMgp is soluble andGPI-cleaved and the other is membrane-bound; (iii) wherein at least oneof the NgR and OMgp is recombinantly expressed on a surface of a cell;etc.

The invention also provides compositions and mixtures specificallytailored for practicing the subject methods. For example, an in vitromixture for use in the subject binding assays comprises NgR, OMgp and anagent, wherein at least one of the NgR and OMgp is soluble andGPI-cleaved. Kits for practicing the disclosed methods may also compriseprinted or electronic instructions describing the applicable subjectmethod.

DESCRIPTION OF PARTICULAR EMBODIMENTS OF THE INVENTION

The following descriptions of particular embodiments and examples areoffered by way of illustration and not by way of limitation. Unlesscontraindicated or noted otherwise, in these descriptions and throughoutthis specification, the terms “a” and “an” mean one or more, the term“or” means and/or and polynucleotide sequences are understood toencompass opposite strands as well as alternative backbones describedherein.

In one embodiment, the invention provides a method for reducing axongrowth inhibition mediated by OMgp and detecting resultant reduced axongrowth inhibition, the method comprising steps: contacting a mixturecomprising an axon and isolated OMgp with an agent and under conditionswherein but for the presence of the agent, the axon is subject to growthinhibition mediated by the OMgp; and detecting resultant reduced axongrowth inhibition, indicating that the agent reduces axon growthinhibition mediated by OMgp.

The recited axons are mammalian neuron axons, preferably adult neuralaxons, which may be peripheral or, preferably CNS neuron axons. Asexemplified below, the method may be applied to neural axons in vitro orin situ.

OMgp is a natural, mammalian CNS myelin glycoprotein (see, Habib et al.1998a, 1998b) which functions as a ligand of the Nogo Receptor (NgR) onCNS axons. OMgp cDNA has been cloned from several species, includinghuman (Genbank Accn No. NM_002544), mouse (Genbank Accn No. NM_019409),and cow (Genbank Accn No. S45673). Note that OMgp cDNA encodes twoalternative initiating methionine residues; compare, Genbank AccessionNos. M63623 (human) and S67043 (mouse). OMgp may be membrane-boundthrough a GPI linkage or cleaved therefrom. As exemplified herein, OMgpmay be obtained on or cleaved from naturally expressing myelin. Also asexemplified herein, OMgp may also be expressed recombinantly in suitablerecombinant expression systems, wherein functional expression may beconfirmed by the growth cone collapsing assays described herein.

The recited isolated OMgp is provided isolated from other components ofOMgp's natural myelin mileau, which may be effected by purification fromsuch components or expression of the OMgp in a non-natural system. Inparticular embodiments, the isolated OMgp is accompanied by othercomponents which provide or interfere with or alter the axon growthinhibitory or NgR binding activity of the OMgp. Preferred isolated OMgpis purified or recombinantly expressed, particularly on a surface of acell.

The recited agent may be characterized as an OMgp-specific binding agentor, particularly as applied to pharmaceutical screens, an agent notpreviously characterized to bind OMgp nor reduce axon growth inhibitionmediated by OMgp, wherein the agent is a candidate agent and thedetecting step characterizes the candidate agent as reducing axon growthinhibition mediated by OMgp. Similarly, the agent may be a candidateagent not previously characterized to reduce axon growth inhibitionmediated by OMgp, wherein the detecting step characterizes the candidateagent as reducing axon growth inhibition mediated by OMgp.

Detailed protocols for implementing the recited steps are exemplifiedbelow and/or otherwise known in the art as guided by the presentdisclosure. The recited contacting and detecting steps are tailored tothe selected system. In vitro systems provide ready access to therecited mixture using routine laboratory methods, whereas in vivosystems, such as intact organisms or regions thereof, typically requiresurgical or pharmacological methods. More detailed such protocols aredescribed below. Similarly, the detecting step is effected by evaluatingdifferent metrics, depending on the selected system. For in vitrobinding assays, these include conventional solid-phase labeled proteinbinding assays, such as ELISA-type formats, solution-phase bindingassays, such as fluorescent polarization or NMR-based assays, etc. Forcell-based or in situ assays, metrics typically involve assays of axongrowth as evaluated by linear measure, density, host mobility or otherfunction improvement, etc.

In another embodiment, the invention provides a method for reducing axongrowth inhibition mediated by OMgp and detecting resultant reduced axongrowth inhibition by (a) contacting a mixture comprising an axon andOMgp with an exogenous OMgp-specific binding agent and under conditionswherein the agent binds the OMgp and but for the presence of the agent,the axon is subject to growth inhibition mediated by the OMgp, and (b)detecting resultant reduced axon growth inhibition.

This protocol may similarly be practiced with in vitro or in vivo,particularly in situ, mixtures. Note that in this embodiment, the agentis necessarily an exogenous OMgp-specific binding agent and the recitedOMgp need not be isolated, i.e. it may be present in the context of itsnative myelin. Accordingly, this aspect of the invention providesmethods for reducing axon growth inhibition mediated by OMgp in itsnative mileau. By reducing axon growth inhibition, the methods assistthe repair of axons following injury or trauma, such as spinal cordinjury. In addition, the methods may be applied to alleviate dysfunctionof the nervous system due to hypertrophy of neurons or their axonalprojections, such as occurs in diabetic neuropathy.

An OMgp-specific binding agent exogenous to an axon or mixturecomprising an axon is not naturally present with the axon or mixture.The OMgp-specific binding agents specifically bind the OMgp of therecited mixture and thereby functionally inhibit the axon collapseand/or NgR binding mediated by the OMgp. Of course, as OMgp-specific,the subject agents inherently do not cross-react with (specifically bindto) structurally distinct NgR ligands, such as NogoA. We haveexemplified suitable OMgp binding agents from diverse structures.Initial agents were identified by selecting high affinity OMgp bindersfrom natural NgR peptides. These assays identified a number ofOMgp-specific NgR peptides encompassing NgR LRR (leucine rich repeat)sequences, including the exemplified species: hNR260/308, mNR260/308 andrNR260/308. Natural OMgp-specific NgR peptide sequences were subject todirected combinatorial mutation and binding analysis. Resultantsynthetic-sequence OMgp-specific peptides include the exemplifiedspecies: s1NGR260/308, s2NR260/308 and s3NR260/308. We also used avariety of OMgp peptide immunogens to generate OMgp-specific antibodiesand antibody fragments, including the exemplified monoclonal antibodiesOM-H2276 and OM-H5831 and the exemplified fragments OMF-H7712 andOMF-H6290. OMgp-specific binding agents are also found in compoundlibraries, including the exemplified commercial fungal extract and asynthetic combinatorial organo-pharmacophore-biased libraries.Structural characterization of the exemplified OMgp binding agents(XR-178892, XR-397344, XR-573632, SY-73273M, SY-32340L and SY-95734E) iseffected by conventional organic analysis.

Of particular interest are size-minimized NgR LRR peptides whicheffectively compete for OMgp ligand binding. We synthesized and screenedlarge libraries of NgR peptides for their ability to bind OMgp andthereby reduce OMgp-mediated axon growth inhibition. This workidentified numerous competitive binding peptides of varying lengthwithin a 49 amino acid region of a NgR C-terminal leucine rich repeat,exemplified with human, mouse and rat repeat sequences (hNR260/308, SEQID NO: 1; mNR260/308, SEQ ID NO:2; and rNR260/308, SEQ ID NO:3).Competitive peptides demonstrating >20% competitive activity comparedwith the source 49 mer are subject to combinatorial mutagenesis togenerate synthetic peptide libraries from which we screen for evenhigher affinity binders. Preferred competitive peptides consist, orconsist essentially of a size-minimized sequence within the disclosedhuman source 49mer, preferably a sequence of fewer than 48, 38, 28 or 18residues, wherein at least 6, 8, 12 or 16 residues are usually requiredfor specific binding. Obtaining additional such native sequence andsynthetic competitive peptides involves only routine peptide synthesisand screening in the disclosed binding and growth assays.

In particular applications, the target cells are injured mammalianneurons in situ, e.g. Schulz M K, et al., Exp Neurol. 1998 February;149(2): 390-397; Guest J D, et al., J Neurosci Res. 1997 Dec. 1; 50(5):888-905; Schwab M E, et al., Spinal Cord. 1997 July; 35(7): 469-473;Tatagiba M, et al., Neurosurg 1997 March; 40(3): 541-546; and Examples,below. For these in situ applications, compositions comprising the OMgpbinding agent may be administered by any effective route compatible withtherapeutic activity of the compositions and patient tolerance. Forexample, for CNS administration, a variety of techniques is availablefor promoting transfer of therapeutic agents across the blood brainbarrier including disruption by surgery or injection, drugs whichtransiently open adhesion contact between CNS vasculature endothelialcells, and compounds which facilitate translocation through such cells.The compositions may also be amenable to direct injection or infusion,intraocular administration, or within/on implants e.g. fibers such ascollagen fibers, in osmotic pumps, grafts comprising appropriatelytransformed cells, etc.

In a particular embodiment, the binding agent is delivered locally andits distribution is restricted. For example, a particular method ofadministration involves coating, embedding or derivatizing fibers, suchas collagen fibers, protein polymers, etc. with therapeutic agents, seealso Otto et al. (1989) J Neurosci Res. 22, 83-91 and Otto and Unsicker(1990) J Neurosc 10, 1912-1921. The amount of binding agent administereddepends on the agent, formulation, route of administration, etc. and isgenerally empirically determined and variations will necessarily occurdepending on the target, the host, and the route of administration, etc.

The compositions may be advantageously used in conjunction with otherneurogenic agents, neurotrophic factors, growth factors,anti-inflammatories, antibiotics etc.; and mixtures thereof, see e.g.Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9^(th)Ed., 1996, McGraw-Hill. Exemplary such other therapeutic agents includeneuroactive agents such as in Table 1.

TABLE 1 Neuroactive agents which may be used in conjunction with OMgpbinding agents. NGF Heregulin Laminin NT3 IL-3 Vitronectin BDNF IL-6Thrombospondin NT4/5 IL-7 Merosin CNTF Neuregulin Tenascin GDNF EGFFibronectin HGF TGFa F-spondin bFGF TGFb1 Netrin-1 LIF TGFb2 Netrin-2IGF-I PDGF BB Semaphorin-III IGH-II PDGF AA L1-Fc Neurturin BMP2 NCAM-FcPercephin BMP7/OP1 KAL-1Abbreviations: NGF, nerve growth factor; NT, neurotrophin; BDNF,brain-derived neurotrophic factor; CNTF, ciliary neurotrophic factor;GDNF, glial-derived neurotrophic factor; HGF, hepatocyte growth factor;FGF, fibroblast growth factor; LIF, leukemia inhibitory factor; IGF,insulin-like growth factor; IL, interleukin; EGF, epidermal growthfactor; TGF, transforming growth factor; PDGF, platelet-derived growthfactor; BMP, bone morphogenic protein; NCAM, neural cell adhesionmolecule.

In particular embodiments, the OMgp binding agent is administered incombination with a pharmaceutically acceptable excipient such as sterilesaline or other medium, gelatin, an oil, etc. to form pharmaceuticallyacceptable compositions. The compositions and/or compounds may beadministered alone or in combination with any convenient carrier,diluent, etc. and such administration may be provided in single ormultiple dosages. Useful carriers include solid, semi-solid or liquidmedia including water and non-toxic organic solvents. As such thecompositions, in pharmaceutically acceptable dosage units or in bulk,may be incorporated into a wide variety of containers, which may beappropriately labeled with a disclosed use application. Dosage units maybe included in a variety of containers including capsules, pills, etc.

The invention also provides pharmaceutical screens for inhibitors ofOmgp-NgR binding, particularly, methods for characterizing an agent asinhibiting binding of NgR to OMgp by: (a) incubating a mixturecomprising NgR, OMgp and an agent under conditions whereby but for thepresence of the agent, the NgR and OMgp exhibit a control binding; and(b) detecting a reduced binding of the NgR to the OMgp, indicating thatthe agent inhibits binding of the NgR to the OMgp.

NgR is a natural, mammalian neural axon protein (Fournier et al., 2001,Nature 409, 341-46) which functions as a receptor for Nogo66 and forOMgp. NgR cDNA has been cloned from several species, including human(Genbank Accn No. BC011787), mouse (Genbank Accn No. NM-022982), and rat(Genbank Accn No. AY028438). NgR may be membrane-bound through a GPIlinkage or cleaved therefrom. As exemplified herein, NgR may be obtainedon or cleaved from naturally expressing myelin. Also as exemplifiedherein, NgR may also be expressed recombinantly in suitable recombinantexpression systems, wherein functional expression may be confirmed bythe growth cone collapsing assays described herein.

The screening method is amenable to a wide variety of differentprotocols. For example, in particular embodiments, at least one of theNgR and OMgp is soluble and GPI-cleaved, one of the NgR and OMgp issoluble and GPI-cleaved and the other is membrane-bound, and at leastone of the NgR and OMgp is recombinantly expressed on a surface of acell.

The invention also provides compositions and mixtures specificallytailored for practicing the subject methods. For example, an in vitromixture for use in the subject binding assays comprises premeasured,discrete and contained amounts of NgR, OMgp and an agent, wherein atleast one of the NgR and OMgp is soluble and GPI-cleaved. Kits forpracticing the disclosed methods may also comprises printed orelectronic instructions describing the applicable subject method.

EXAMPLES

OMgp is a Physiological Nogo Receptor Ligand and Inhibitor of NeuriteOutgrowth. To examine whether any GPI-linked proteins in CNS myelin mayact as inhibitors of neurite outgrowth, we treated purified bovine whitematter myelin with phosphatidylinositol-specific phospholipase C(PI-PLC) and examined the released proteins for their ability to altergrowth cone morphology in a growth cone collapse assay using embryonicday 13 chick dorsal root ganglia (E13 DRG)^(6,9,10). We found thatPI-PLC-released CNS myelin proteins, when added to the DRG culturemedium, exhibited potent growth cone collapsing activity. To furthercharacterize the inhibitory activity in the PI-PLC-released proteins, weanalyzed solubilized proteins by SDS-PAGE and silver staining and foundthat a band of approximately 110 kDa in size was significantly enrichedin this fraction. Since the size of this band was similar to that of apreviously identified CNS myelin protein OMgp^(11,12), we usedantibodies specific for OMgp to detect enrichment of cleaved OMgp in thePI-PLC supernatants by Western blot. Anti-OMgp antibodies detected aband of comparable size in the PI-PLC-treated supernatants, indicatingthat OMgp is a component released from CNS myelin by PI-PLC. Next, weexamined whether purified recombinant OMgp protein was able to act as aninhibitor of neurite outgrowth in two well-established in vitro assays,the growth cone collapse^(6,9,13) and the neurite outgrowthassays^(3-7,9). Similar to the PI-PLC-treated myelin supernatants,purified recombinant polyhistidine-tagged OMgp protein (OM-his), but notproteins purified from control vector-transfected COS-7 cells, inducedthe collapse of growth cones derived from E13 chick DRG neurons in adose-dependent manner. The effective concentration for half-maximalresponse (EC₅₀) for OM-his was approximately 1.5 nM. Consistent with itbeing an inhibitor of neurite outgrowth, the GPI-anchored OMgp proteinwas found to be highly expressed by myelin basic protein (MBP)-positivemature oligodendrocytes and enriched in the axon-adjacent myelinlayers^(11,12). Moreover, OM-his, when presented either as animmobilized substrate or in a soluble form, inhibited neurite outgrowthof cerebellar granule neurons (CGN) from postnatal day 7-9 (P7-9) rats,also in a dose-dependent manner. The OMgp-induced inhibitory responseswere similar to that brought about by treatment of the same neurons withan alkaline phosphatase (AP) fusion protein containing the 66 amino acidextracellular domain of Nogo-A (AP-66)^(6,9), indicating that OMgp, likeNogo-66, is a potent neurite outgrowth inhibitor.

To further examine the functional importance of OMgp as a CNS myelinassociated inhibitor, we used peanut agglutinin (PNA)-Agarose beads¹¹ tospecifically deplete OMgp, but not Nogo-A or MAG, fromβ-octylglucoside-solublized CNS myelin. We found that while theinhibitory activity in the PNA-depleted myelin was significantly reducedcompared to control myelin, the OMgp-enriched eluates from the same PNAcolumn displayed a potent inhibitory activity. We next compared therelative contributions of OMgp with two other known inhibitors, MAG andNogo-A, to the inhibitory activity associated with CNS Myelin. Anionexchange chromatography was able to biochemically separate MAG andNogo-A^(3,15), but failed to separate OMgp from Nogo-A. Thus, we appliedthe OMgp-depleted PNA column flow-through myelin fractions onto aQ-Sepharose column and generated fractions enriched in MAG or Nogo-A bysequential elution of the column with increasing concentrations of NaCl.Our data revealed that the fractions enriched in MAG or Nogo-A, similarto the OMgp-enriched eluates from the PNA column, all significantlyinhibited neurite outgrowth. Comparisons of the EC₅₀ for each of thesefractions allowed us to estimate that the OMgp-enriched fractioninhibited neurite outgrowth to a similar extent as the Nogo-A-enrichedfraction, but much stronger than the MAG-enriched-fraction. Our resultis consistent with previous observations that the major inhibitoryactivity of CNS myelin resides in the 0.30 to 0.5 M NaCl elutionfractions of Sepharose Q columns^(15,16), as the majority of OMgpco-fractionates with Nogo-A in this chromatography procedure.

To investigate the mechanisms by which OMgp inhibits neurite outgrowth,we used an expression cloning strategy^(9,13,16) to identify cellsurface OMgp-binding proteins. An AP fusion protein containing OMgp(AP-OM) was able to bind to the surface of rat P9 CGNs and to inducecollapse of E13 chicken DRG growth cones, and thus was used in the cellsurface binding assays. From pools of an adult human brain cDNAexpression library, we isolated two cDNAs that encoded OMgp-bindingproteins. Sequence analysis revealed that both cDNAs contained thefull-length coding region of the Nogo-66 receptor (NgR), which had beenpreviously identified as a high-affinity receptor for the extracellulardomain of Nogo-A⁹. We then established a CHO cell line stably expressingthe NgR and determined the binding affinity of expressed NgR for AP-OMas 5 nM, similar to what had been determined for Nogo-66 (7 nM, ref. 9).These data indicate that NgR is a high-affinity OMgp-binding protein. Wenext performed co-precipitation experiments by incubating GST or a GSTfusion protein containing the entire extracellular domain of NgR(GST-NgR) with OM-his alone or in the presence of AP or AP-66 protein.We found that GST-NgR, but not the control GST protein, bound to OM-his,indicating a direct interaction between NgR and OMgp. We next determinedwhich domain(s) of OMgp was responsible for binding to NgR. Like NgR,OMgp is also a GPI-linked protein containing a leucine-rich repeat (LRR)domain. An AP fusion protein containing only the LRR domain of OMgp(AP-LRR) was found to be sufficient to bind strongly to NgR-expressingcells. In addition, the C-terminal domain with serine-threonine repeats(AP-S/T) alone was also able to bind, though less strongly, to NgRexpressing cells.

To further determine how NgR interacted at the molecular level withthese two inhibitors, we made a series of deletion constructs of NgR andfound that both the LRR and the C-terminal LRR (LRRCT) domains of NgRwere required for highest binding to OMgp and that OMgp and Nogo-66appear to bind overlapping regions of NgR. Consistently, in both cellsurface binding and the co-precipitation assay, AP-66 and OM-hisproteins competed for binding to NgR. To examine the functionalconsequences of the molecular interaction of the two ligands with NgR,we compared the collapsing activity of OM-his plus AP-66 with that ofOM-his or AP-66 alone. The estimated EC₅₀ for OM-his plus Nogo-66 (2.5nM) was similar to that of OM-his (1.5 nM) and AP-66 (2.3 nM),indicating an additive effect between OM-his and AP-66 in inducinggrowth cone collapse. As the binding affinities of both OMgp and Nogo-66to NgR are similar, our results together imply that these two myelincomponents act independently through NgR to inhibit neurite outgrowth.

As the GPI-linked NgR protein can be released by PI-PLC, we nextexamined whether PI-PLC treatment could affect axonal responsiveness toOMgp. Consistent with a previous study⁹, PI-PLC treatment did not alterthe growth cone morphology of E13 chick DRG neurons, but rendered theseaxons insensitive to Nogo-66. Similarly, PI-PLC treatment also abolishedthe growth cone-collapsing activity of OMgp. As a control, the growthcone collapsing activity of Semaphorin 3A (Sema 3A)^(10,13), known to bemediated by transmembrane receptor molecules including neuropilin-1 andmembers of the plexin family¹⁸, was not affected by PI-PLC treatment.Even though PI-PLC also cleaves other GPI-anchored proteins on theaxonal surface, these results indicated that GPI-anchored proteins, suchas NgR, act as necessary signal transducers of the inhibitory activityof OMgp.

To assess whether NgR is capable of mediating OMgp-induced inhibitoryactivity on neurite outgrowth, we next took a gain-of-function approachto examine whether expression of NgR was able to conferOMgp-responsiveness to otherwise insensitive neurons. It has been shownpreviously that chick E7 retinal ganglion neurons (RGN) are insensitiveto Nogo-66, but that introduction of exogenous NgR in these neuronsrendered their growth cones to be responsive to Nogo-66⁹. Using the samestrategy, we made a recombinant herpes simplex virus (HSV) that drivesexpression of a FLAG-tagged full-length human NgR (FLAG-NgR) in infectedneurons. Upon infection, 80% of the E7 RGNs expressed the FLAG-NgRprotein as assessed by immunocytochemistry with an anti-FLAG antibody.No significant morphological changes were observed in the HSV-infectedneurons. Consistent with a previous study⁹, expression of FLAG-NgRconferred a growth cone collapse response to Nogo-66 in E7 RGNs.Furthermore, the growth cones of NgR-expressing axons also becomecollapsible by OMgp. In contrast, a control virus driving the expressionof β-galactosidase did not alter the axonal responses of the sameneurons to either Nogo-66 or OMgp. Taken together, our results indicatethat, like Nogo-66, OMgp acts through NgR and its associated receptorcomplex to inhibit axon outgrowth. As opposed to Nogo-A, the majority ofwhich is localized intracellularly⁵⁻⁷, OMgp is predominantly localizedon the surfaces of oligodendrocytes and axon-adjacent myelinlayers^(11,12,14), indicating that OMgp is a physiological ligand ofNgR.

Purification, PI-PLC Treatment, and OMgp Depletion of Myelin. Myelin wasprepared from white matter of bovine brain according to establishedprotocols¹⁹. In brief, white matter tissues were homogenized in 0.32 Msucrose in phosphate-buffered saline (PBS) and the crude myelin thatbanded at the interphase of a discontinuous sucrose gradient (0.32M/0.85 M) was collected and purified by two rounds of osmotic shock withdistilled water and re-isolation over the sucrose gradient. For PI-PLCtreatment, aliquots of myelin suspensions in water (10 mg/ml) wereincubated with or without 2.5 U/ml PI-PLC (Sigma) at 37° C. for 2 hr,prior to centrifugation (360,000 g for 60 min). The supernatants wereconcentrated, partitioned in Triton X-114, and used for assays and fordetection with Western analysis.

To deplete OMgp, myelin was first solublized with 1% octylglucoside andthe resultant extract was passed twice through columns with PNA-Agarose(Vector Laboratories) or control Agarose beads as describedpreviously¹². The OMgp-enriched fraction was obtained by eluting thePNA-Agarose column with buffer containing 0.5 M D-galactose. As asignificant portion of OMgp co-fractionated with Nogo-A in anionexchange columns¹², we enriched MAG or Nogo-A from myelin by applyingthe flow-through fractions from the PNA-Agarose columns onto aQ-Sepharose (Sigma) column¹⁵. The column was then eluted stepwise withequal amount of buffers containing 0.15 M (MAG-enriched), 0.45 M (Nogo-Aenriched), or 1.0 M NaCl¹⁵. Aliquots of individual fractions were testedfor their inhibitory activity in the neurite outgrowth assay asdescribed previously^(15,16,21).

Expression Cloning and Binding Experiments. Sequences encoding mouseOMgp were amplified from Marathon-ready mouse cDNA (Clontech) andconfirmed by sequencing analysis, prior to subcloning into theexpression vector AP-5⁹ for expressing an AP-OM fusion protein taggedwith both a polyhistidine and a myc epitope. The resultant plasmid DNAwas transfected into COS-7 cells and the secreted protein purified usingnickel-Agarose resins (Qiagen).

Cell surface binding and expression cloning were performed as describedpreviously^(4,13,17). To detect AP-OM binding, cultures were washed withbinding buffer (Hanks balanced salt solution containing 20 mM Hepes, pH7.5, and 1 mg/ml bovine serum albumin). The plates were then incubatedwith AP-OM-containing binding buffer for 75 min at room temperature.After extensive washing and heat inactivation, the bound proteins weredetected by AP staining using nitro blue tetrazolium (NBT) and5-bromo-4-chloro-3-indoyl phosphate (BCIP) as substrate. For saturationanalysis, we disrupted cells and detected bound AP fusion proteins usingr-nitrophenyl phosphate as substrate.

For expression cloning of OMgp-binding proteins, pools of 5,000 arrayedclones from a human brain cDNA library (Origene Technologies, Rockville,Md.) were transfected into COS-7 cells, and AP-OM binding was assessedas above. We isolated single NgR cDNA clones by sub-dividing the poolsand sequencing analysis.

Generation of Recombinant Proteins and Viruses and Co-precipitation. Toexpress recombinant OMgp for function assays, we subcloned the codingregion of mouse OMgp (amino acids 23-392) into pSecTag B (Invitrogen) toexpress his-tagged OMgp protein (OM-his) in COS-7 cells. The expressedOM-his protein was purified using a nickel resin. To constructrecombinant herpes simplex viruses (HSV), cDNAs for FLAG-tagged NgR orb-galatosidase were inserted into the HSV amplicon HSV-PrpUC andpackaged into the virus using helper 5dl1.2, as described previously²⁰.The resultant viruses were purified on sucrose gradients, pelleted, andresuspended in 10% sucrose. The titer of the viral stocks was 4.0×10⁷infectious units/ml. For each study, aliquots from the same batches ofviral vectors were used. In order to produce recombinant Nogo-66protein, the sequence of Nogo-66 was amplified from a human cDNA clone,KIAA0886, from the Kazusa DNA Research Institute and used to generate aconstruct expressing the AP-66 protein as described by GrandPre et al⁶.Antibodies against Nogo-A and MAG were purchased from Alpha Diagosticsand R & D Systems, respectively.

In co-precipitation experiments, 2 mg GST or GST-NgR were firstimmobilized to glutathione-Agarose beads and the beads were furtherincubated with or without 1 mg OM-his in the presence of 2 mg of AP orAP-66 at 4° C. for 2 hr. After extensive washing, the bound proteinswere resolved with SDS-PAGE and detected by Western blotting.

Growth Cone Collapse and Neurite Outgrowth Assays. Chick E13 DRG and E7retina were isolated and cultured as described previously^(9,10). DRGexplants cultured overnight were used for growth cone collapse assays.To assess the effects of PI-PLC treatment, cultures were pre-incubatedwith 2 U/ml PI-PLC for 30 min prior to treatment with individual testproteins for an additional 30 min. To express NgR in E7 retinal ganglionneurons, we infected the explants with recombinant HSV for 24 hr. Afterincubation with each test protein for 30 min, retinal explants werefixed in 4% paraformaldehyde and 15% sucrose. Infection of HSV-LacZ wasdetected by a standard b-galatosidase staining protocol²⁰. FLAG-NgRexpression was detected by incubating paraformaldehyde-fixed cultureswith M2 anti-FLAG antibody (Sigma). Bound antibody was detected byincubation with AP-conjugated anti-rabbit IgG second antibody andreaction with NBT and BCIP (Vector labs). Growth cone collapse wasquantified only in those positively stained for b-galatosidase orimmunoreactive for the FLAG epitope.

Neurite outgrowth assays were performed as described previously^(15,21).Briefly, P7-9 rat CGNs were dissected and then plated at a density of1×10⁵ cells per well. The cells were cultured for 24 hr prior tofixation with 4% paraformaldehyde and staining with a neuronal specificanti-b-tubulin antibody (TuJ-1, Covance). Quantification of neuritelength and statistical analysis were performed as describedpreviously²².

Oligodendrocyte precursor cells were isolated from the cerebralhemispheres of P1 rats and differentiated in vitro as described³.Immunostaining of mature oligodendrocytes was performed using antibodiesagainst MBP (Sigma) and OMgp¹⁴.

Exemplary OMgp Binding (OMgp-NgR Binding Inhibitory) Agents. An AP-OMgpfusion protein, prepared as described above, was used to evaluate theOMgp binding affinity of a variety of candidate binding agents asmeasured by the ability of agents preincubated with OMgp to inhibitsubsequent OMgp-NgR binding. The selected binding assay formats areguided by structural requirements of the candidate agents and includeCOS-expression, solid phase ELISA-type assay, and fluorescentpolarization assays. Candidate agents were selected from natural andsynthetic peptide libraries biased to natural NgR LRR (supra) sequences,OMgp-specific monoclonal antibody (Mab) and Mab fragment libraries, acommercial fungal extract library, and a synthetic combinatorialorgano-pharmacophore-biased library. In each instance, we assay specificbinding inferentially by evaluating the affect of preincubating the OMgpwith the agent, on subsequent OMgp-NgR binding. Selected exemplary highaffinity OMgp-specific binding agents subject to in vivo activity assays(below) are shown in Table 2.

TABLE 2 Selected exemplary high-affinity OMgp-specific binding agents;(u), structure not yet determined. Sequence/ Binding OMgp Binding AgentClass/Source Structure Assay 1. hNR260/308 natural peptide SEQ ID NO: 1++++ 2. mNR260/308 natural peptide SEQ ID NO: 2 ++++ 3. rNR260/308natural peptide SEQ ID NO: 3 ++++ 4. s1NR260/308 synthetic peptide SEQID NO: 4 ++++ 5. s2NR260/308 synthetic peptide SEQ ID NO: 5 ++++ 6.s3NR260/308 synthetic peptide SEQ ID NO: 6 ++++ 7. OM-H2276 monoclonalantibody IgG ++++ 8. OM-H5831 monoclonal antibody IgG ++++ 9. OMF-H7712Fab fragment (Mab) IgG Fab2 ++++ 10. OMF-H6290 Fab fragment (Mab) IgGFab2 ++++ 11. XR-178892 fungal extract library natural (u) ++++ 12.XR-397344 fungal extract library natural (u) ++++ 13. XR-573632 fungalextract library natural (u) ++++ 14. SY-73273M combinatorial librarysynthetic (u) ++++ 15. SY-32340L combinatorial library synthetic (u)++++ 16. SY-95734E combinatorial library synthetic (u) ++++

Corticospinal Tract (CST) Regeneration Assay. High affinity OMgp bindingagents demonstrating inhibition of OMgp-mediated in vitro axon growthcone collapse as described above are assayed for their ability toimprove corticospinal tract (CST) regeneration following thoracic spinalcord injury by promoting CST regeneration into human Schwann cell graftsin the methods of Guest et al. (1997, supra). For these data, the humangrafts are placed to span a midthoracic spinal cord transection in theadult nude rat, a xenograft tolerant strain. OMgp binding agentsdetermined to be effective in in vitro collapse assays are incorporatedinto a fibrin glue and placed in the same region. Anterograde tracingfrom the motor cortex using the dextran amine tracers, Fluororuby (FR)and biotinylated dextran amine (BDA), are performed. Thirty-five daysafter grafting, the CST response is evaluated qualitatively by lookingfor regenerated CST fibers in or beyond grafts and quantitatively byconstructing camera lucida composites to determine the sprouting index(SI), the position of the maximum termination density (MTD) rostral tothe GFAP-defined host/graft interface, and the longitudinal spread (LS)of bulbous end terminals. The latter two measures provide informationabout axonal die-back. In control animals (graft only), the CST do notenter the graft and undergo axonal die-back. As shown in Table 3, theexemplified binding agents dramatically reduce axonal die-back and causesprouting and these in vivo data are consistent with the correspondinggrowth cone collapsing activity.

TABLE 3 In Vitro and Vivo Neuronal Regeneration with Exemplery OMgpBinding Agents. Collapse Reduced Promote OMgp Binding Agent InhibitionDie-Back Sprouting 1. hNR260/308 ++++ ++++ ++++ 2. mNR260/308 ++++ ++++++++ 3. rNR260/308 +++ +++ +++ 4. s1NR260/308 ++++ ++++ ++++ 5.s2NR260/308 +++ +++ +++ 6. s3NR260/308 ++++ ++++ ++++ 7. OM-H2276 ++++++++ ++++ 8. OM-H5831 ++++ ++++ ++++ 9. OMF-H7712 +++ +++ +++ 10.OMF-H6290 +++ +++ +++ 11. XR-178892 +++ +++ +++ 12. XR-397344 ++++ ++++++++ 13. XR-573632 ++++ ++++ ++++ 14. SY-73273M +++ +++ +++ 15.SY-32340L ++++ ++++ ++++ 16. SY-95734E +++ +++ +++

Peripheral Nerve Regeneration Assay. High affinity OMgp binding agentsdemonstrating inhibition of OMgp-mediated in vitro axon growth conecollapse as described above are also incorporated in the implantabledevices described in U.S. Pat. No. 5,656,605 and tested for thepromotion of in vivo regeneration of peripheral nerves. Prior tosurgery, 18 mm surgical-grade silicon rubber tubes (I.D. 1.5 mm) areprepared with or without guiding filaments (four 10-0 monofilamentnylon) and filled with test compositions comprising the binding agentsof Table 2. Experimental groups consist of: 1. Guiding tubes plusBiomatrix 1T™ (Biomedical Technologies, Inc., Stoughton, Mass.); 2.Guiding tubes plus Biomatrix plus filaments; 3-23. Guiding tubes plusBiomatrix 1™ plus binding agents.

The sciatic nerves of rats are sharply transected at mid-thigh and guidetubes containing the test substances with and without guiding filamentssutured over distances of approximately 2 mm to the end of the nerves.In each experiment, the other end of the guide tube is left open. Thismodel simulates a severe nerve injury in which no contact with thedistal end of the nerve is present. After four weeks, the distance ofregeneration of axons within the guide tube is tested in the survivinganimals using a functional pinch test. In this test, the guide tube ispinched with fine forceps to mechanically stimulate sensory axons.Testing is initiated at the distal end of the guide tube and advancedproximally until muscular contractions are noted in the lightlyanesthetized animal. The distance from the proximal nerve transectionpoint is the parameter measured. For histological analysis, the guidetube containing the regenerated nerve is preserved with a fixative.Cross sections are prepared at a point approximately 7 mm from thetransection site. The diameter of the regenerated nerve and the numberof myelinated axons observable at this point are used as parameters forcomparison.

Measurements of the distance of nerve regeneration document therapeuticefficacy. Similarly, plots of the diameter of the regenerated nervemeasured at a distance of 7 mm into the guide tube as a function of thepresence or absence of one or more binding agents demonstrate a similartherapeutic effect of all 16 tested. No detectable nerve growth ismeasured at the point sampled in the guide tube with the matrix-formingmaterial alone. The presence of guiding filaments plus thematrix-forming material (no agents) induces only very minimalregeneration at the 7 mm measurement point, whereas dramatic results, asassessed by the diameter of the regenerating nerve, are produced by thedevice which consisted of the guide tube, guiding filaments and bindingagent compositions. Finally, treatments using guide tubes comprisingeither a matrix-forming material alone, or a matrix-forming material inthe presence of guiding filaments, result in no measured growth ofmyelinated axons. In contrast, treatments using a device comprisingguide tubes, guiding filaments, and matrix containing binding agentscompositions consistently result in axon regeneration, with the measurednumber of axons being increased markedly by the presence of guidingfilaments.

OMgp-Specific Monoclonal Antibodies Promote Axon Regeneration In Vivo.In these experiments, our OM-H2276 and OM-H58310 Mgp-specific monoclonalantibodies are shown to promote axonal regeneration in the rat spinalcord. Tumors producing our OMgp-specific antibodies, implantationprotocols and experimental design are substantially as used for IN-1 asdescribed in Schnell et al., Nature 1990 Jan. 18; 343(6255):269-72. Inbrief, our OM-H2276 and OM-H5831 monoclonal antibodies are appliedintracerebrally to young rats by implanting antibody-producing tumours.In 2-6-week-old rats we make complete transections of the corticospinaltract, a major fibre tract of the spinal cord, the axons of whichoriginate in the motor and sensory neocortex. Previous studies haveshown a complete absence of cortico-spinal tract regeneration after thefirst postnatal week in rats, and in adult hamsters and cats. In ourtreated rats, significant sprouting occurs at the lesion site, and fineaxons and fascicles can be observed up to 7-11 mm caudal to the lesionwithin 2-3 weeks. In control rats, a similar sprouting reaction occurs,but the maximal distance of elongation rarely exceeded 1 mm. Theseresults demonstrate the capacity for CNS axons to regenerate andelongate within differentiated CNS tissue after neutralization ofOMgp-mediated axon growth inhibition.

OMgp-Specific Monoclonal Antibody Fragments Promote Axon Regeneration inVivo. In these experiments, OMgp-specific monoclonal antibody fragmentsare shown to promote sprouting of Purkinje cell axons. Experimentalprotocols were adapted from Buffo et al., 2000, J Neuroscience 20,2275-2286.

Animals and surgical procedures. Adult Wistar rats (Charles River,Calco, Italy) are deeply anesthetized by means of intraperitonealadministration of a mixture of ketamine (100 mg/kg, Ketalar; Bayer,Leverkusen, Germany) and xylazine (5 mg/kg, Rompun; Bayer).

Fab fragment or antibody injections are performed as previouslydescribed (Zagrebelsky et al., 1998). Animals are placed in astereotaxic apparatus, and the dorsal cerebellar vermis exposed bydrilling a small hole on the posterosuperior aspect of the occipitalbone. The meninges are left intact except for the small hole produced bythe injection pipette penetration. In test rats a recombinant Fabfragment of the OM-H2276 and OM-H5831 antibodies (produced in E. coli),which neutralizes OMgp-associated axon growth cone collapse in vitro isinjected into the cerebellar parenchyma. Three 1 μl injections of Fabfragments in saline solution (5 mg/ml) are performed 0.5-1 mm deep alongthe cerebellar midline into the dorsal vermis (lobules V-VII). Theinjections are made by means of a glass micropipette connected to aPV800 Pneumatic Picopump (WPI, New Haven, Conn.). The frequency andduration of pressure pulses are adjusted to inject 1 μl of the solutionduring a period of ˜10 min. The pipette is left in situ for 5 additionalminutesto avoid an excessive leakage of the injected solution. As acontrol, an affinity-purified F(ab′)₂ fragment of a mouse anti-human IgG(Jackson ImmunoResearch Laboratories, West Grove, Pa.) is applied toanother set of control rats using the same procedure. Survival times forthese two experimental sets are 2, 5, 7 and 30 d (four animals for eachtime point). An additional set of intact animals are examined asuntreated controls.

Histological procedures. At different survival times after surgery,under deep general anesthesia (as above), the rats are transcardiallyperfused with 1 ml of 4% paraformaldehyde in 0.12 M phosphate buffer, pH7.2-7.4. The brains are immediately dissected, stored overnight in thesame fixative at 4° C., and finally transferred in 30% sucrose in 0.12 Mphosphate buffer at 4° C. until they sink. The cerebella are cut using afreezing microtome in several series of 30-μm-thick sagittal sections.One series is processed for NADPH diaphorase histochemistry. Thesesections are incubated for 3-4 hr in darkness at 37° C. in a solutioncomposed of -NADPH (1 mg/ml, Sigma, St. Louis, Mo.) and nitrobluetetrazolium (0.2 mg/ml, Sigma) in 0.12 M phosphate buffer with 0.25%Triton X-100. In some cases (two animals per treated and control sets at2 and 5 d survival), microglia are stained by incubating one sectionseries with biotinylated Griffonia simplicifolia isolectin B4 [1:100 inphosphate buffer with 0.25% Triton X-100; Sigma (Rossi et al., 1994a)]overnight at 4° C. Sections are subsequently incubated for 30 min in theavidin-biotin-peroxidase complex (Vectastain, ABC Elite kit, Vector,Burlingame, Calif.) and revealed using the 3,3′ diaminobenzidine (0.03%in Tris HCl) as a chromogen.

All of the other series are first incubated in 0.3% H₂O₂ in PBS toquench endogenous peroxidase. Then, they are incubated for 30 min atroom temperature and overnight at 4° C. with different primaryantibodies: anti-calbindin D-28K (monoclonal, 1:5000, Swant, Bellinzona,Switzerland), to visualize Purkinje cells; anti-c-Jun (polyclonal,1:1000, Santa Cruz Biotechnology, Santa Cruz, Calif.); and anti-CD11b/c(monoclonal OX-42, 1:2000, Cedarlane Laboratories, Homby, Ontario) tostain microglia. All of the antibodies are diluted in PBS with 0.25%Triton X-100 added with either normal horse serum or normal goat serumdepending on the species of the second antibody. Immunohistochemicalstaining is performed according to the avidin-biotin-peroxidase method(Vectastain, ABC Elite kit, Vector) and revealed using the 3,3′diaminobenzidine (0.03% in Tris HCl) as a chromogen. The reactedsections are mounted on chrome-alum gelatinized slides, air-dried,dehydrated, and coverslipped.

Quantitative analysis. Quantification of reactive Purkinje cells in thedifferent experiments is made by estimating the neurons labeled by c-Junantibodies as previously described (Zagrebelsky et al., 1998). For eachanimal, three immunolabeled sections are chosen. Only vermal sectionsclose to the cerebellar midline that contain the injection sites areconsidered. The outline of the selected sections is reproduced using theNeurolucida software (MicroBrightField, Colchester, Vt.) connected to anE-800 Nikon microscope, and the position of every single-labeled cellcarefully marked. The number of labeled cells present in the threereproduced sections is averaged to calculate values for every individualanimal, which are used for statistical analysis carried out by Student'stest.

A morphometric analysis of Purkinje axons in the different experimentalconditions for each animal, is performed using threeanti-calbindin-immunolabeled sections, contiguous to those examined forc-Jun, as described in Buffo et al. (supra). Morphometric measurementsare made on 200×250 μm areas of the granular layer chosen bysuperimposing a grid of this size on the section. The selected areasencompass most of the granular layer depth and contain only minimalportions of Purkinje cell layer or axial white matter. In each of theselected sections is sampled one area from the dorsal cortical lobulesand one from the ventral cortical lobules. In addition, to sample fromthe different parts of these two cortical regions, areas from differentlobules are selected in the three sections belonging to each individualanimal, one area in each of lobules V, VI, and VII and one in lobules I,II, and IX. All of the anti-calbindin-immunolabeled Purkinje axonsegments contained within the selected areas are reproduced using theNeurolucida software (MicroBrightField) connected to an E-800 Nikonmicroscope with 20× objective, corresponding to 750× magnification onthe computer screen. Each labeled axon segment or branch is reproducedas a single profile. From these reproductions the software calculatesthe number of axon profiles, their individual length, and the totallength of all the reproduced segments, the mean profile length (totallength/number of profiles), and the number of times that the axonscrossed a 25×25 μm grid superimposed on the selected area. Datacalculated from the different areas in the three sections sampled fromeach cerebellum are averaged to obtain values for every individualanimal. Statistical analysis is performed on the latter values (n=4 forall groups at all time points) by Student's t test and paired t test.

Our results reveal significant promotion of sprouting of Purkinje cellaxons in test rats subject to our OM-H2276 and OM-H58310 Mgp-specificmonoclonal antibody fragments as compared with the control animals.

FOOTNOTED REFERENCES

-   1. Schwab, M. E., and Bartholdi, D. Degeneration and regeneration of    axons in the lesioned spinal cord. Physiol. Rev. 76, 319-370 (1996).-   2. Horner, P. J. Gage, F. H. Regenerating the damaged central    nervous system. Nature 407, 963-970 (2000).-   3. McKerracher, L et al., Identification of myelin-associated    glycoprotein as a major myelin-derived inhibitor of neurite growth.    Neuron 13, 805-811 (1994).-   4. Mukhopadhyay, G. et al., A novel role for myelin-associated    glycoprotein as an inhibitor of axonal regeneration. Neuron 13,    757-767 (1994).-   5. Chen, M. S. et al., Nogo-A is a myelin-associated neurite    outgrowth inhibitor and an antigen for monoclonal antibody IN-1.    Nature 403, 434-439 (2000).-   6. GrandPre, T., Nakamura, F., Vartanian, T., Strittmatter, S. M.    Identification of the Nogo inhibitor of axon regeneration as a    Reticulon protein. Nature 403, 439-444 (2000).-   7. Prinjha, R. et al., Inhibitor of neurite outgrowth in humans.    Nature 403, 383-384 (2000).-   8. Tessier-Lavigne, M, Goodman, C. S. Perspectives: neurobiology.    Regeneration in the Nogo zone. Science 287, 813-814 (2000).-   9. Fournier, A. E., GrandPre, T., Strittmatter, S. M. Identification    of a receptor mediating Nogo-66 inhibition of axonal regeneration.    Nature 409, 341-346 (2001); see also, Strittmatter, US2002/0012965.-   10. Luo, Y., Raible, D., Raper, J. A. Collapsin: a protein in brain    that induces the collapse and paralysis of neuronal growth cones.    Cell 75, 217-227 (1993).-   11. Mikol, D. D., Stefansson, K. A phosphatidylinositol-linked    peanut agglutinin-binding glycoprotein in central nervous system    myelin and on oligodendrocytes. J Cell Biol 106, 1273-1279 (1988).-   12. Mikol, D. D., Gulcher, J. R., Stefansson, K. The    oligodendrocyte-myelin glycoprotein belongs to a distinct family of    proteins and contains the HNK-1 carbohydrate. J Cell Biol 110,    471-479 (1990).-   13. He, Z, and Tessier-Lavigne, M. Neuropilin is a receptor for the    axonal chemorepellent Semaphorin III. Cell 90, 739-751, 1997.-   14. Habib, A. A., Marton L. S., Allwardt, B., Gulcher, J. R.,    Mikol, D. D., Hognason, T., Chattopadhyay, N., Stefansson, K.    Expression of the oligodendrocyte-myelin glycoprotein by neurons in    the mouse central nervous system. J Neurochem 70, 1704-1711 (1998).-   15. Niederost, B. P., Zimmermann, D. R., Schwab, M. E.,    Bandtlow, C. E. Bovine CNS myelin contains neurite growth-inhibitory    activity associated with chondroitin sulfate proteoglycans. J    Neurosci 19, 8979-8989 (1999).-   16. Spillmann, A. A., Bandtlow, C. E., Lottspeich, F., Keller, F.,    Schwab, M. E Identification and characterization of a bovine neurite    growth inhibitor (bNI-220). J Biol Chem 273, 19283-19293 (1998).-   17. Flanagan, J. G. and Cheng, H.J. Alkaline phosphatase fusion    proteins for molecular characterization and cloning of receptors and    their ligands. Methods Enzymol 327, 198-210 (2000).-   18. Liu B. P, Strittmatter S. M. Semaphorin-mediated axonal guidance    via Rho-related G proteins. Curr Opin Cell Biol 13, 619-626 (2001).-   19. Norton, W. T., and Poduslo, S. E. Myelination in rat brain:    method of myelin isolation. J. Neurochem. 21, 749-757 (1973).-   20. Neve, R. L, Howe, J. R, Hong, S, Kalb, R. G. Introduction of the    glutamate receptor subunit 1 into motor neurons in vitro and in vivo    using recombinant herpes simplex virus. Neuroscience 79, 435-447    (1997).-   21. Huang, D. W., McKerracher, L., Braun, P. E., David, S. A    therapeutic vaccine approach to stimulate axon regeneration in the    adult mammalian spinal cord. Neuron 24, 639-647 (1999).-   22. Cohen-Cory, S, and Fraser, S. E. Effects of brain-derived    neurotrophic factor on optic axon branching and remodeling in vivo.    Nature 378, 192-196 (1995).-   23. Oka, A., Belliveau, M. J., Rosenberg, P. A., and Volpe, J. J.    Vulnerability of oligodendroglia to glutamate: Pharmacology,    mechanisms and prevention. J. Neurosci. 13, 1441-1453 (1993).-   24. Takahashi, T., Nakamura, F., Jin, Z., Kalb, R. G.,    Strittmatter, S. M. Semaphorins A and E act as antagonists of    neuropilin-1 and agonists of neuropilin-2 receptors. Nat Neurosci 1,    487-493 (1998).

The foregoing descriptions of particular embodiments and examples areoffered by way of illustration and not by way of limitation. Allpublications and patent applications cited in this specification and allreferences cited therein are herein incorporated by reference as if eachindividual publication or patent application or reference werespecifically and individually indicated to be incorporated by reference.Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to those of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

1. A method for characterizing an agent as inhibiting binding of NgR(Nogo receptor) to OMgp (oligodendrocyte-myelin glycoprotein), themethod comprising the steps of: incubating a mixture comprising NgR,OMgp and an agent under conditions whereby but for the presence of theagent, the NgR and OMgp exhibit a control binding; and specificallydetecting a reduced binding of the NgR to the OMgp, indicating that theagent inhibits binding of the NgR to the OMgp.
 2. A method according toclaim 1, wherein at least one of the NgR and OMgp is soluble.
 3. Amethod according to claim 1, wherein one of the NgR and OMgp is solubleand the other is membrane-bound.
 4. A method according to claim 1,wherein at least one of the NgR and OMgp is recombinantly expressed on asurface of a cell.
 5. A method according to claim 1 wherein the agent ispreincubated with the OMgp to inhibit subsequent OMgp-NgR binding.
 6. Amethod according to claim 1 wherein the method comprises a formatselected from the group consisting of COS-expression, solid phaseELISA-type assay, and fluorescent polarization assay.
 7. A methodaccording to claim 1 wherein the agent is selected from natural andsynthetic peptide libraries biased to natural NgR LRR sequences,OMgp-specific monoclonal antibody (Mab) and Mab fragment libraries, acommercial fungal extract library, and a synthetic combinatorialorgano-pharmacophore-biased library.
 8. A method according to claim 1wherein the method comprises a corticospinal tract (CST) regenerationassay format.
 9. A method according to claim 1 wherein the methodcomprises a peripheral nerve regeneration assay format.